Vitamin K-dependent proteins are used to treat certain types of hemophilia. Classic hemophilia or hemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation Factor VIII, and affects almost exclusively males with an incidence between one and two individuals per 10.000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with Factor VII concentrates was introduced the mean life span for a person with severe hemophilia was less than 20 years. The use of concentrates of Factor VIII from plasma and later on that of recombinant forms of FVIII has considerably improved the situation for the hemophilia patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. Hemophilia B being 5 times less prevalent than hemophilia A is caused by non-functional or missing FIX and is treated with FIX concentrates from plasma or a recombinant form of FIX. In both hemophilia A and in hemophilia B the most serious medical problem in treating the disease is the generation of alloantibodies against the replacement factors. Up to 30% of all hemophilia A patients develop antibodies to FVIII. Antibodies to FIX occur to a lesser extent but with more severe consequences, as they are less susceptible to immune tolerance induction therapy.
The current model of coagulation states that the physiological trigger of coagulation is the formation of a complex between tissue factor (TF) and Factor VIIa (FVIIa) on the surface of TF expressing cells which are normally located outside the vasculature. This leads to the activation of FIX and FX ultimately generating some thrombin. In a positive feedback loop thrombin activates FVIII and FIX, the so-called “intrinsic” arm of the blood coagulation cascade, thus amplifying the generation of FXa, which is necessary for the generation of the full thrombin burst to achieve complete hemostasis. It was shown that by administering supraphysiological concentrations of FVIIa hemostasis is achieved bypassing the need for FVIIa and FIXa. The cloning of the cDNA for FVII (U.S. Pat. No. 4,784,950) made it possible to develop a recombinant replacement of that plasma derived coagulation factor. This FVIIa was successfully administered for the first time in 1988 to a patient with a high titer of inhibitory antibodies to FVIII. Ever since the number of indications of FVIIa has grown steadily showing a potential to become a universal hemostatic agent (Erhardtsen, 2002).
FVII is a single-chain glycoprotein with a molecular weight of 50 kDa, which is secreted by liver cells into the blood stream as an inactive zymogen of 406 amino acids. It contains 10 γ-carboxy-glutamic acid residues (positions 6, 7, 14, 16, 19, 20, 25, 26, 29, and 35) localized in the Gla-domain of the protein. The Gla residues require vitamin K for their biosynthesis. Located C-terminal to the Gla domain are two epidermal growth factor domains followed by a trypsin-type serine protease domain. Further posttranslational modifications of FVII encompass hydroxylation (Asp 63), N-(Asn145 and Asn322) as well as O-type glycosylation (Ser52 and Ser60).
FVII is converted to its active form FVIIa by proteolysis of the single peptide bond at Arg152-Ile153 leading to the formation of two polypeptide chains, a N-terminal light chain (17 kDa) and a C-terminal heavy chain (28 kDa) which are held together by one disulfide bridge. In contrast to other vitamin K-dependent polypeptides no activation peptide which is cleaved off during activation has been described for FVII. The Arg152-Ile153 cleavage site corresponds by homology comparison to the C-terminal activation cleavage site of other vitamin K-dependent polypeptides. However as Arg144 might also constitute a protease cleavage site it cannot be excluded that FVII in contrast to current thinking possesses an activation peptide of 8 amino acids between Arg144 and Arg152.
Essential for attaining the active conformation of FVIIa is the formation of a salt bridge after activation cleavage between Ile153 and Asp343. Activation of FVII can be achieved in vitro by FXa, FXIIa, FIXa, FVIIa, FSAP and thrombin. Mollerup et al. (Biotechnol. Bioeng. (1995) 48: 501-505) reported that some cleavage also occurs in the heavy chain at Arg290 and or Arg315.
FVII is present in plasma in a concentration of 500 ng/ml. 1%, e.g. 5 ng/ml of FVII is present as FVIIa. Plasma half-life of FVII was found to be about 4 hours and that of FVIa about 2 hours. Although the half-life of FVIIa of 2 hours is comparatively long for an activated coagulation factor, which is, otherwise more in the order of minutes due to the irreversible inhibition by Serpins like antithrombin III, this nevertheless constitutes a severe drawback for the therapeutic use of FVIIa, as it leads to the need of multiple i.v. injections or continuous infusion to achieve hemostasis resulting in very high cost of treatment and inconvenience for the patient. As on the other hand FVIIa has the potential to be used as a universal hemostatic agent there is a high medical need to develop forms of FVIIa which have a longer functional half-life.
Several attempts have been made to modify FVII:
Nicolaisen et al. (WO 88/10295, Jun. 25, 1987) suggest that by deleting or modifying the following amino acids FVII will be stabilized against proteolytic degradation: Lys32, Lys38, Lys143, Arg290, Arg315, Lys316, Lys341, Arg392, Arg 396, Arg 402, Ile42 and Tyr44.
Nicolaison (U.S. Pat. No. 5,580,560, Nov. 13, 1989) extends WO 88/10295 to include also mutations or deletions in Arg304, Phe278 and Tyr332 to render FVII/FVIIa less susceptible to proteolysis.
Bharadwaj et al. (JBC (1996), 48 pp. 30685-30691) expressed the FVII mutant Phe328Ser that failed to activate FX and showed no detectable amidolytic activity. Dickinson et al. (PNAS (1996) 93, 14379-14384) proposed FVIIa variants in which Lys157, Val158, Glu296, Met298, Asp334, Ser336 or Lys337 have been replaced by Ala.
Nelsestuen (WO 99/29767 Oct. 23, 1997) modified the Gla domain by introducing point mutations in a way to enhance its affinity to phospholipid membranes thereby resulting into a modified FVIIa with enhanced specific activity. Proposed point mutations are at Pro10, Gly11, Arg28 and Lys32.
Nelsestuen (WO 00/66753, Apr. 29, 1999) modified the Gla domain by introducing point mutations in a way to enhance its affinity to phospholipid membranes thereby resulting into a modified FVIIa with enhanced specific activity. Proposed point mutations are at 5, 9, 11, 12, 29, 33, 34, 35 and/or 36.
Kornfelt et al. (Archiv. Biochem. and Biophys., 363, pp 43-54) showed that the oxidation of Met298 and Met306 leads to a 30% higher dissociation rate of FVIIa-ox from TF and a 20% lower FX activation as compared to wild type FVIIa.
Kemball-Cook et al. (JBC (1998), 14 pp. 8516-8521) expressed the FVII mutant Gln100Arg and showed that it had no detectable clotting activity though having amidolytic activity comparable to wild type FVIIa and speculate that this might be due to impaired association with TF.
Iino et al. Arch. Biochem. Biophys. (1998) 352:182-192 showed that mutating the O-glycosylation sites Ser-52 and Ser-60 decreases the coagulatory activity of FVIIa possibly interfering with the interaction with TF.
Ruf et al. (Biochemistry (1999) 16, pp. 1957-66) showed that the mutation Arg36Ala leads to decreased rate of FX activation.
Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466 abstract 1474) refer to a FVII variant in which residues 316-320 are deleted or residues 311-322 are replaced with the corresponding residues from trypsin.
Soeiima Kenji et al. (JP2001061479, Aug. 24, 1999) created a modified FVIIa with enhanced specific activity by cleaving the disulfide group between Cys159 and Cys164 or by substituting, adding or deleting at least a part of the loop structure from Thr233 to Asp244 or by substituting, adding, or deleting at least a part of the intervening sequence between Arg304 and Cys329.
Pedersen et al. (US 2003/0096338 Feb. 11, 2000) claim conjugates of FVII and FVIIa with non-polypeptidic moieties including also sugars with the aim to prolong FVIIa half-life. The claims also encompass the introduction of novel N- and/or O-type glycosylation sites or the introduction of novel combined with the removal of a preexisting N- and/or O-type glycosylation sites to obtain in vivo glycoconjugates.
Persson and Olsen (US 2003/0170863, May 3, 2000) taught modified FVIIa in which Leu305 or Phe374 have been replaced by another amino acid. At most 20 amino acids in the protease domain (153-406) have been replaced in combination with the above mentioned mutations. Other modified FVII molecules are disclosed which have optionally other amino acids replaced in positions 274, 300-304 and 306-312 in combination with Leu305 and Phe374. These modifications have the effect that FVIIa will spontaneously attain a more active conformation that normally has to be induced by TF.
Persson and Olsen (US 2003/0104978 and 2003/0100740, Sep. 29, 2000) further taught other modified FVIIa molecules with point mutations other than Ala substitutions at positions Lys157, Lys337, Asp334, Ser336, Val158, Glu296 and Met298.
Pingel and Klausen (US 2002/0151471 and US 2002/0137673, Oct. 2, 2000) claim a preparation comprising a plurality of FVII or related polypeptides, which comprise certain ratios of different N-type glycosylations.
Ruf et al. (WO 02/38162, Nov. 9, 2000) claimed FVII/FVIIa variants with the modifications Met298Gln, Glu296Ile and Val158Asn or combinations thereof leading to a higher amidolytic activity in the absence of TF and a higher affinity to TF. The factor was further modified to increase its stability in modifying the trypsin-like cleavage sites at Lys32, Lys38, Arg290, Arg304, Arg315 and Lys341 and the chymotrypsin-like sites at Ile42, Tyr44, Phe278 and Tyr332.
Persson (WO 02/077218, Mar. 22, 2001) teaches FVII/FVIIa mutants in which amino acids 247-260, 393-405 and Pro406 are mutated, more specifically R396, Q250 and Pro406, preferably an amino acid to which a chemical group can be attached with the goal of increasing the half life of FVII/FVIIa. This can be combined with mutations which increase the activity of FVII/FVIIa at K157, V158, E296, M298, L305, D334, S336, K337 and F374.
Persson and Olsen (US 2003/0100075, Sep. 27, 2001) teach that Leu305 is located at the end of an α-Helix found in the TF complexed form of FVIIa, which is believed to be important for the activity. In free FVIIa this helix is distorted and thus possibly unstable. Replacing Leu305 with other amino acids leads according to this invention to variants which attain the active conformation which otherwise is induced by TF. The amino acids Lys157, Lys337, Asp334, Ser336, Val 158, Glu296 and Met298 are located in areas which affect the formation of the salt bridge between Ile153 and Asp343. Replacing these amino acids leads according to this invention to the facilitation of the insertion of the N-terminus of the protease e.g. the generation of the salt bridge essential for activity.
Persson and Olsen (US 2003/0130191, Nov. 2, 2001) teach further modified FVII/VIIa mutants with increased specific activity which are substituted with other amino acids in positions: 313-329, 364, 366, 373 and 376 as well as in positions 330-339.
Haaning et al. (WO 03/093465, Apr. 30, 2002) extend the teaching of Nelsestuen (modification of the Gla Domain to enhance phospholipid binding), namely a substitution at Pro10 preferably Gln, Lys32 preferably Glu, Asp33 preferably a hydrophobic amino acid preferably Phe, Ala34 preferably a negatively charged amino acid preferably Glu and an insertion of an amino acid after Ala3 preferably Tyr with the introduction of further N-glycosylation sites.
Foncuberta et al. (WO 2004/011675, Jul. 25, 2002) describe naturally occurring allelic variants of FVII which could theoretically lead to higher expression levels and improved function of FVIIa. No data for such improved properties are shown. Two variants out of 49 were found in exons and lead to a substitution of amino acids: A294V and R353Q.
Persson and Olsen (WO 2004/029090, Sep. 25, 2002) showed that mutating Phe374 in combination with some other amino acids leads to an increase of TF independent activity of FVIIa. namely L305V, S314E, K337A and F374Y led to an increase of the TF amidolytic activity.
Haaning et al. (WO 2004/029091, Sep. 30, 2002) modified FVII at L39, I42, S43, K62, L65, F71, E82 and F275 in the TF binding site of FVII/FVIIa increasing the affinity to TF.
Andersen et al. (WO 2004/083361, Mar. 20, 2003) modified FVII/FVIIa in positions 196 (D196N/K), 237 (G237L or insertions GM GAAA or GAAA) and 341 (K341N/Q) to increase affinity to TF.
Blaichman et al. (WO 2004/108763, Jun. 5, 2003) modified FVII/FVIIa within the EGF domain based on an analysis of differences between the human and rabbit EGF domain as rabbit Factor VIIa has higher affinity to human TF as human Factor VIIa. Mutants in position 53, 62, 74, 75 and 83 are claimed and shown to have higher affinity to human TF and increased hemostatic potential.
Haaning et al (WO 2004/111242, Jun. 19, 2003) modified FVII/FVIIa at: positions 4, 10, 28, 32, 33, 34, 36, 74, 77, 116 preferably A3Y, P10Q, R28F, K32E, D33F, A34L, R36E, K38E, P74S, E77A, E116D. The R36E mutation causes reduced binding to TF and reduced thrombin generation in TF-dependent assays while maintaining in PL-dependent assays the same activity.
Johansen et al. (WO 2005/032581, Oct. 7, 2003) Teaches hybrid molecules consisting of a lipid membrane binding domain coupled to a Factor VII activity domain optionally coupled to a bulking agent, preferentially to PEG.
Maun et al. Protein Sci. (2005) 14:1171-80 introduced new disulfide bonds to lock the FVII conformation into an active TF*FVIIa-like state. Kinetic analysis of amidolytic activity revealed that all Factor VIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Factor VIIa disulfide-locked variants no longer required TF as a co-factor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type.